H1FOO-DD promotes efficiency and uniformity in reprogramming to naive pluripotency

Summary Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.

Figure S1 related to Figure 1.Characterization of H1FOO-DD vector (A).PCA of RNA-seq data from primed human PSCs in this study compared to human PSCs.(B).Number of genes with MAE >2.0 among the clones in each group in RNA-seq.(C).GO terms for the 1350 genes with MAE >2.0 genes among the OSKL-iPSC clones.(D).Number of probes with MAE >2.0 among the clones in each group in DNA methylation array.(E).Schematic representation of the trilineage differentiation and analysis protocol.(F).Dot plot of algorithmic scores generated by Scorecard analysis based on 96 genes expression per sample.n=1 of each point.(G).Representative immunofluorescent staining for ACTININ and TNNT2 of cardiomyocytes at day 10 post differentiation from OSKLH-iPSCs.Scale bar, 100 μm.(H).Quantification of TNNT2 expression in primed OSKL-iPSC and OSKLH-iPSC by flow cytometry.(I).Percentage of TNNT2 expressed cells by flow cytometry.n=6 of each clone.

Figure S2 related to Figure 2 .
Figure S2 related to Figure 2. Specific gene expression in each cell group and clusters in single cell RNA-seq analysis (A).HDF, PSC and OSKL or HDF, PSC and OSKLH plotted UMAPs for comparing the difference in distribution between OSKL and OSKLH.(B).Heatmap of top 10 representative marker genes expression in each cluster.(C).Feature plot of naive pluripotency, epithelial and mesoendoderm related gene expression in the UMAP.

Figure S3 related to Figure 3 .
Figure S3 related to Figure 3. Specific gene expression in each cell group and clusters in single cell RNA-seq analysis (A).Percentage of reads counted within the detected peak region (peak±500 bp) in HDF, SeV-OSKL or SeV-OSKLH infected HDF and PSC.(B).HDF, PSC and OSKL or HDF, PSC and OSKLH plotted UMAPs for comparing the difference in distribution between OSKL and OSKLH.(C).Gene activity plot of pluripotency markers L1TD1, NANOG, LIN28 and DPPA4.LIN28 and DPPA4 are known to be expressed from the late reprogramming stage.(D).Plot of clusters produced by single cell RNA-seq on the single cell ATAC-seq UMAP by matching the gene activity data of single cell ATAC-seq and the gene expression data of single cell RNA-seq.Dashed arrows indicate the reprogramming process inferred to be followed by the majority of SeV-OSKL or SeV-OSKLH-infected HDFs.(E).Percentage of cells in each cell group in single cell ATAC-seq that matched the gene expression patterns of the clusters created by single cell RNA-seq analysis.

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Figure S4 related to Figure 4. Exploration of factors that play an important role in modified reprogramming by H1FOO-DD (A).Representative DEGs detected in the bulk RNA-seq analysis.(B).Number of primed and naive human iPSC colonies generated from HDFs at day 14.Data are shown as the mean ± s.d.n=3.*P<0.05.(C).Coverage plot of FKBP1A coding region in OSKL and OSKLH at day 2 and day 5 obtained by single cell ATAC-seq.(D).Dot plot of FKBP1A expression in OSKL and OSKLH in the UMAP obtained by single cell RNA-seq.(E).Feature plot of FKBP1A expression in OSKL and OSKLH in the UMAP obtained by single cell RNA-seq.(F).Feature plot of H1FOO expression in OSKL and OSKLH in the UMAP obtained by single cell RNA-seq.(G).Protein expression analysis of phosphorylated TGFBR1 (pTGFBR1) and TGFBR1by Western blotting.We quantified the expression level of pTGFBR1 with TGFBR1 protein expression.Data are shown as the mean ± s.d.n=3.***P<0.001.

Figure S5 related to Figure 6 .Figure
Figure S5 related to Figure 6.Exploration of factors that play an important role in modifiedreprogramming by H1FOO-DD (A).PCA of RNA-seq data from primed and naive human PSCs in this study compared to reset naive human iPSCs and pre-implantation embryo samples from(Takashima et al., 2014;Theunissen et al., 2014;Yan et al., 2013).(B) Heatmap of the RNA-seq data depicting expression levels of shared, primed, and naive